Hemophilia A, the most common of the severe, inherited bleeding disorders, results from a deficiency or defect in the plasma protein, factor VIII. There is no cure for Hemophilia A and treatment consists of replacement therapy using preparations of the (purified) plasma or recombinant protein.
Factor VIII circulates as an inactive, procofactor form in complex with von Willebrand factor, which stabilizes factor VIII and potentially helps to localize it to sites of vascular injury. Proteolytic activation of factor VIII releases the active cofactor form of the protein, factor VIIIa, facilitating its association in the intrinsic factor Xase complex. This complex, consisting of the serine protease factor IXa and factor VIIIa assembled on an anionic phospholipid membrane surface catalyzes the conversion of factor X to factor Xa, an essential reaction for the propagation phase of blood coagulation. The role of factor VIIIa is to increase the catalytic efficiency of factor IXa by several orders of magnitude (Fay, “Activation of Factor VIII and Mechanisms of Cofactor Action,” Blood Rev. 18:1-15 (2004)).
Following generation of the fibrin clot, components of the clotting cascade is shut down by a variety of mechanisms. The down regulation of factor Xase occurs by two mechanisms, both of which involve the cofactor, factor VIIIa. One mechanism involves the dissociation of a critical subunit of factor VIIIa, the A2 subunit that exists in a weak affinity interaction with the other subunits of factor VIIIa. The second mechanism occurs by limited proteolysis of factor VIIIa and is catalyzed by the anti-coagulant protease, activated protein C (“APC”). The relative contributions of these two mechanisms to the inactivation of factor VIIIa and subsequent down regulation of factor Xase are not fully understood, although both components are thought to be important in vivo (Fay, “Activation of Factor VIII and Mechanisms of Cofactor Action,” Blood Rev. 18:1-15 (2004)).
Significant interest exists in stabilizing factor VIIIa activity, since a more “inactivation-resistant” form of the protein would represent a superior therapeutic for hemophilia A, potentially requiring less material to treat the patient (Fay et al., “Mutating Factor VIII: Lessons from Structure to Function,” Blood Reviews 19:15-27 (2005)). To this end, preparations of factor VIII have been described where mutations have been made in the recombinant protein to prevent the dissociation of the A2 subunit by introducing novel covalent bonds between A2 and other factor VIII domains (Pipe et al., “Characterization of a Genetically Engineered Inactivation-resistant Coagulation Factor VIIIa,” Proc Natl Acad Sci USA 94:11851-11856 (1997); Gale et al., “An Engineered Interdomain Disulfide Bond Stabilizes Human Blood Coagulation Factor VIIIa,” J. Thrombosis & Haemostasis 1: 1966-1971 (2003)). In addition, the sites of APC-catalyzed proteolysis (cleavage) in factor VIII are known (Fay, “Activation of Factor VIII and Mechanisms of Cofactor Action,” Blood Rev. 18:1-15 (2004)) and occur at P1 arginine (Arg) residues at positions 336 and 562. (Residues surrounding the cleavage site are indicated as: HN2-P4-P3-P2-P1-P1′-P2′-P3′-COOH relative to the scissile bond at P1-P1′. This is otherwise known as the P4-P3′ region, which extends from residues 333-339 of SEQ ID NO: 2.) Cleavage at each site, i.e., Arg336 and Arg562, contributes to the inactivation of factor VIIIa. Furthermore, cleavage at either site is independent of the other, and the former site appears to be the more reactive site as the rate of cleavage at Arg336 is ˜25-fold more rapid than cleavage at Arg562 in factor VIIIa (Varfaj et al., “Role of P1 Residues Arg336 and Arg562 in the Activated-Protein-C-catalysed Inactivation of Factor VIIIa,” Biochem. J. 396:355-362 (2006)). Thus, attack at Arg336 is the dominant site for APC inactivation of factor VIIIa activity. Prior studies have shown that replacing the P1 Arg residues with other amino acids such as leucine or glutamine by site-directed mutagenesis yield cleavage-resistant forms of factor VIII that are not inactivated by the APC-dependent pathway (Amano et al., “Mutation at either Arg336 or Arg562 in Factor VIII is Insufficient for Complete Resistance to Activated Protein C (APC)-mediated Inactivation: Implications for the APC Resistance Test,” Thrombosis & Haemostasis 79:557-563 (1998)). This is due to the inability of substrate to correctly dock at the APC active site if the P1 residue is not Arg.
It has since been determined that neither of these types of mutation is desirable in a therapeutic factor VIII, because they substantially eliminate means for down-regulation. This situation could yield a prothrombotic condition, which may cause harm. Thus, a more desirable situation would be to reduce the rate of factor VIIIa inactivation by the APC pathway rather than substantially eliminate this pathway for factor VIIIa inactivation.
The present invention is directed to overcoming these and other deficiencies in the art.